In Gene Roddenberry's first television production, the 1963-64 NBC series The Lieutenant, Koenig played a significant role as noncom Sgt. John Delwyn, who is recommended for Officer Candidates School by the series protagonist, Lt William T. (Tiberius) Rice, played by Gary Lockwood; (in episode 27, "Mother Enemy", aired on 4 April 1964). The plot twist, at the height of the US-Soviet Cold War, is that Sgt Delwyn's visiting mother is a prominent, and politically active, American Communist Party member. This sets up various interesting plot tensions involving Delwyn, Rice, and Rice's CO, Capt. Rambridge, played by Robert Vaughn.

I was only one of two people who auditioned for the part, which is quite extraordinary. Considering that this has so materially affected the last 35 years of my life ... a couple of hours after I auditioned I heard that I had gotten the role.

Koenig played Ensign Pavel Chekov, navigator on the USS Enterprise, in the original Star Trek television series (starting in Season 2) and in all of the films featuring the original cast (including Star Trek: Generations). One of only two actors to audition, he was cast as Chekov almost immediately primarily because of his resemblance to British actor/musician Davy Jones of the Monkees. Show creator Gene Roddenberry hoped that Koenig would increase the show's appeal to young people.[5][6] As the 30-year old's hair was already receding, costume designers fashioned a Davy Jones-style "moptop" hairpiece for him. In later episodes, his own hair grew out enough to accomplish the look with a comb-over.[7] (The studio's publicity department, however, falsely ascribed the inclusion of Chekov to an article in Pravda that complained about the lack of Russians in Star Trek.[5]) Roddenberry asked him to "ham up" his Russian accent to add a note of comic relief to the series. Chekov's accent has been criticized as inauthentic, in particular Koenig's substituting the "w" sound in place of a "v" sound (e.g., "wodka" for "vodka"); Koenig has said the accent was inspired by his father, who had the same difficulty with the "v" sound.[4]

Most of Koenig's fan mail indeed came from children, and the high volume of letters contributed to him soon receiving a contract as a regular cast member; this surprised Koenig, who had been told that Chekov would be a recurring role.[5][6] When the early Season 2 episodes of Star Trek were shot, George Takei (who played Sulu) was delayed while completing the movie The Green Berets, so Chekov is joined at the Enterprise helm by a different character. When Takei returned, the two had to share a dressing room and a single episode script. This reportedly angered Takei to the point where he nearly left the show, but the two actors have since become good friends, to the point that Koenig was the best man at Takei's wedding in 2008.[8] The image of the two characters at the helm and navigation stations of the Enterprise became iconic.[9]

Koenig's Chekov character never appeared in the animated Star Trek for budgetary reasons; however, he is credited with writing an episode of that series titled "The Infinite Vulcan", making him the first cast member to write a Star Trek story for television.

After Chekov, Koenig had a recurring role as Psi CopAlfred Bester on the television series Babylon 5. He was a "Special Guest Star" in twelve episodes and, at the end of the third season, the production company applied for an Emmy nomination on his behalf. He was slated to play Bester on the spin-off series Crusade, but the series was cancelled before his episode was filmed. The character name of "Alfred Bester" was an homage to the science-fiction writer of the same name.

Koenig's film, stage, and TV roles span fifty years. He has played roles ranging from a teenage gang leader (Alfred Hitchcock Presents) to Scandinavian fiance Gunnar in the Gidget episode entitled "Gidget's Foreign Policy": COMBAT! s.1 ep.12: "The Prisoner" (1962) Minor role as soldier on guard duty, to a Las Vegas entertainer (I Spy), and a minor role in the TV series Mannix, in a 1968 episode called "Delayed Action". He also had a short role in the 1976 Columbo episode "Fade in to Murder". He returned to space with a starring role in Moontrap and played a futuristic dictator in the video game Maximum Surge.[10] The game was later cancelled, but considerable footage from it was recycled for the film Game Over, with Koenig's dialogue dubbed over in order to retrofit his performance into the role of a computer hard drive. During the early 90's he starred in a touring production of the play "The Boys in Autumn", playing a middle aged Tom Sawyer, who reunites with childhood friend Huckleberry Finn. Fellow Trek actor Mark Lenard played Finn. In addition to acting, he has written several films (Actor, I Wish I May, You're Never Alone When You're a Schizophrenic), one-act plays, and a handful of episodes for TV shows: Star Trek: The Animated Series, Land of the Lost, Family and The Powers of Matthew Star. He has also written several books, including Warped Factors: A Neurotic's Guide to the Universe (an autobiography), Chekov's Enterprise (a journal kept during the filming of Star Trek: The Motion Picture) and Buck Alice and the Actor-Robot (a science fiction novel), which was re-released in 2006. He created his own comic book series called Raver, which was published by Malibu Comics in the early 1990s, and appeared as a "special guest star" in an issue of the comic book Eternity Smith, which features him prominently on its cover.[11] In 2013 he released the graphic novel "Walter Koenig's Things To Come" with artist J.C. Baez, published by Bluewater Comics, which compiled the 4 issues of the mini-series of the same name.[12]

In 1989, Koenig starred in the science fiction film Moontrap as Mission commander Colonel Jason Grant.

In 1997, Koenig starred in Drawing Down the Moon, an independent film about a Wiccan woman who attempts to open a homeless shelter in a small Pennsylvania town. Koenig played Joe Merchant, a local crime lord obsessed with chaos theory who sends his thugs to intimidate her into shutting down the shelter.

In 2004, Koenig co-starred in Mad Cowgirl, an independent movie about a meat-packing health inspector dying from a brain disorder in which he played televangelist Pastor Dylan. The movie played the SF Indiefest and the Silverlake Film Festival, followed by a limited release in major cities. Mad Cowgirl was released on DVD on December 5, 2006.

In 2007, Koenig was asked by the human rights group U.S. Campaign for Burma to help in their grassroots campaign about the humanitarian crisis in Burma. As detailed on his official website, he visited refugee camps along the Burma-Thailand border from July 16 to July 25, 2007.